PROJECT SUMMARY Abscisic acid (ABA) is a central stress hormone in Arabidopsis that down-regulates cell proliferation and causes cell cycle arrest. Early signal transduction networks that down-regulate cell proliferation are of key importance for controlling mitogenesis and their mis-regulation is linked to many human diseases. The long- term goal of this research is to achieve a quantitative understanding of the network of events that mediate early abscisic acid signaling making use of the potent Arabidopsis guard cell system. We will characterize newly identified key cellular signaling mechanisms hypothesized to control the early ABA signaling core consisting of ABA receptors, PP2C protein phosphatases and SnRK2 & calcium (Ca2+)-dependent protein kinases. Aims: I. Direct regulatory proteins of eukaryotic PP2C phosphatase activity are not well-understood. We will characterize the functions of a newly identified early PP2C control loop that can mediate ABA receptor-PP2C signaling robustness. This loop includes the PP2C interacting GDP/GTP exchange factor 1 (GEF1), which activates ROP10 and ROP11, which in turn stabilize PP2C activity via mechanisms that will be elucidated. Moreover, PP2Cs protect GEF1 and Ca2+-dependent protein kinases (CPKs) cause ABA-mediated GEF1 degradation via unknown mechanisms. ABA regulation of this newly identified control loop will be functionally dissected and the role of this loop in mediating Ca2+ specificity during ABA signaling will be characterized by biochemical, genetic, biophysical, in vivo time-resolved, guard cell signaling and structural analyses. II. Previous studies have shown that activation of the PP2C down-regulated OST1-SnRK2 protein kinases, which orchestrate downstream ABA signaling, requires phosphorylation of the SnRK2 kinase activation loop. However, whether this activation of OST1-SnRK2 kinases is accomplished by auto-phosphorylation or by other protein kinases has remained unclear. Our recent results show that dephosphorylated OST1 cannot re-activate itself. Via a genetic redundancy screen, we have identified MAP-kinases (MAPKKKs) that activate OST1, in contrast to several other ABA signaling kinases tested. We will investigate the hypothesis that these MAPKKKs represent the long-sought activation mechanism of OST1-SnRK2 protein kinases in the ABA signaling core. III. Through our recent development of an innovative genome-wide artificial microRNA screening platform, a systems biological approach will be pursued that enables screening for key (partially) redundant genes that function in the ABA signaling network. Newly isolated and confirmed mutants with altered abscisic acid responses will be characterized through a combination of systems biology, genetics, and functional analyses towards characterizing their direct roles in the dynamic ABA signaling network. This research will result in a new understanding of PP2C phosphatase and SnRK2 kinase regulation and Ca2+ specificity signaling mechanisms, which are fundamental to numerous cell signaling processes and disease states, and will reveal novel mechanisms in the early ABA signaling core.